Avermectins and milbemycins to treat parasitic infestations in dogs

ABSTRACT

Avermectins and milbemycins to treat endo- and ectoparasitic infestations in dogs.

This application is a 371 of PCT/GB91/01981 filed Nov. 11, 1991.

This invention relates to a method of treating parasites in dogs.

This invention is particularly concerned with the use of certainavermectins and milbemycins described in EP-A-0 421 568 (U.S. Ser. No.525,094) for the treatment or prophylaxis of endo- and ectoparasiticinfestations of dogs.

The compounds used in this invention have parasiticidal properties, forexample against nematodes, and are useful for the treatment ofhelminthiasis.

The term helminthiasis encompasses diseases of animals caused byinfestation with parasitic worms such as Strongyles, Ascarids,hookworms, lungworms, filarial worms and whipworms.

The compounds used in this invention are also active against Arthropods.The phylum Arthropoda comprises insects--such as biting flies, lice,bugs, beetles and fleas--and arachnids--such as mites and ticks.

Thus the present invention provides the use of a compound of generalformula (I) as defined below for the manufacture of a medicament for thetreatment or prophylaxis of endo and ectoparasitic infestations,especially helminthiasis and arthropod or nematode infestations, indogs.

The present invention also provides a method of treatment or prophylaxisof endo- and ectoparasitic infestations, especially helminthiasis andanthropod or nematode infestations, which comprises administering aneffective non-toxic amount of a compound of general formula (I) asdefined below to a dog in need thereof.

This invention is particularly concerned with the use of a compound ofgeneral formula (I) to combat round worms, hook worms, whipworms anddemodectic mange in dogs. As indicated above, the invention alsoincludes prophylatic treatment against parasites, especially againstheartworm, by killing the larval stage.

For use according to the invention the compounds of general formula (I)may be formulated for administration with carriers and adjuvants in anyconvenient way for use in veterinary medicine, by analogy with knownanthelmintics.

In suitable formulations the compounds of general formula (I) may beadministered to animals orally (as a paste, drench, bolus, capsule ortablet), parenterally, percutaneously, as a food additive (eg granules,pellets or powder), topically (as a cream, wash or spray), ortransdermally (as a pour-on).

The compounds of general formula (I) may be formulated as a mixture witheach other and/or with other anthelmintics, insecticides, acaricides orother pharmacologically active substances.

Suitably the composition consists of sufficient material to provide adose of from 0,001 to 100mg of active ingredient per kg of animal bodyweight per dose, more suitably 0.01 to 10mg/kg per dose.

A composition for use in the invention may suitably contain from 0.1% byweight, preferably from 1.0 to 60% by weight, of the compound of generalformula (I) (based on the total weight of the composition), depending onthe method of administration.

It will be appreciated that, in some cases, it will be advisable torepeat the dosing of the infected or potentially infected animal withthe compound of general formula (I) according to conventional dosageregimes used with anthelmintics:

The compound used in this invention is an avermectin or milbemycinselected from avermectins and milbemycins having the general formula(I): ##STR1## wherein R¹ is hydrogen or optionally protected hydroxy; R²is alkoxy, optionally protected hydroxy, oxo or optionally 0-substitutedoximino; R³ is hydrogen, optionally protected hydroxy,or a group4'-(α-L-oleandrosyl)-α-L-oleandrosyloxy or α-L- oleandrosyloxy whereinthe terminal hydroxy group is optionally protected; R⁴, R⁵, R⁶ and R⁷are the same or different and each is hydrogen or an organic radical;and R⁸ is an optionally substituted amine or imino group such asoptionally 0-substituted oxyimino, optionally N-substituted hydrazone,or optionally N-substituted semicarbazone; with the proviso that thecompound of formula (I) is not a compound of formula (E) or (F) below ora compound disclosed in EP-A-0 307 220. Typically R³ is in theα-configuration. When R³ is in the β-configuration then it is preferablyoptionally protected hydroxy, and/or R¹ is preferably hydrogen, and/orR² is preferably methoxy or optionally protected hydroxy.

EP-A-0 259 779, EP-A-0 293 549, EP-A-0 307 225 and GB-A-2192630 describecompounds of formula (E): ##STR2## wherein R¹ is optionally protectedhydroxy or methoxy, ^(Rm) is optionally protected hydroxy, oxo, or animino group such as optionally 0-substituted oxyimino, optionallyN-substituted hydrazone, or optionally N-substituted semicarbazone,R^(n) is methyl, ethyl or isopropyl, and the dashed line is a doublebond or an epoxide group.

EP-A-0 260 536 and EP-A-0 260 537 describe compounds of formula (F):##STR3## wherein R^(o) is optionally protected hydroxy or methoxy, ^(Rp)is hydrogen or a sugar residue, R^(q) is optionally protected hydroxy,oxo, or an imino group such as optionally 0-substituted oxyimino oroptionally N-substituted hydrazone, and R^(r) is isopropyl or sec-butyl.

The above compounds of general formula (I) form the subject of EP-A-0421 568 (U.S. patent application Ser. No. 525,094 filed May 17, 1990,now abandoned) which also describes methods of their preparation andindicates preferred substitutents.

A corresponding disclosure exists in the following numbered patentapplications of the countries indicated:

Australia 55140/90; Canada 2017030; Eire 1785/90; Japan 125638/90;Mexico 21897; New Zealand 233680; Portugal 94075; South Africa 90/3703;South Korea 7054/90; Taiwan (ROC) 79104620.

The disclosure of the above-mentioned patent applications isincorporated herein by reference.

Preferably the compound used in the present invention is VS 54396 or VS55759 disclosed respectively in Example 6 (Z- and E-isomers) and inExample 36 (E-isomer) of EP-A-0 421 568 (U.S. patent application Ser.No. 525,094 filed May 17, 1990; now abandoned). The preparation of thesecompounds is illustrated below as Examples 1 and 2.

REFERENCE EXAMPLE 23 Oxo-25 (S)-t-butyl milbemycin x

To a solution of (4S)-5,5-dimethyl-4-triethylsilyloxy-1-hexyne (8.3 g,33 mmol) in THF (100 ml) at -78° C. under a nitrogen atmosphere wasadded butyllithium (1.6M in hexane, 18.9 ml, 30 mmol) dropwise over aperiod of 5 mins. and the mixture stirred at -78° C. for a further 3 H.A solution of VS 48927 prepared as described in Examples 1 to 3 ofEP-A-0319142 (4.8 g, 8.6 mmol) in THF (20 ml) was added to the mixturewhich was stirred at -78° C. for a further 15 mins. The reaction wasquenched with a cold solution (˜-20° C.) of glacial acetic acid (10 ml)in THF (10 ml) and the mixture was then allowed to warm to 0° C. Brine(100 ml) was added and the mixture was extracted with ether (3×100 ml).The combined organic extracts were washed with water, dried (MgSO₄) andevaporated to an approximate volume of 50 ml. Methanol (50 ml) was addedand the solution again evaporated to an approximate volume of 50 ml.4-Toluenesulphonic acid (1 g) was added and the mixture stirred at 20°C. for 1 h.

Sodium bicarbonate (100 ml) was added to the mixture and the wholeextracted with dichloromethane (3×100 ml). The combined organic extractswere washed with brine (100 ml), dried (MgSO₄) and evaporated. Theresidue was purified by column chromatography (silica eluted initiallywith dichloromethane and subsequently gradient eluted with 10-60% ethylacetate in hexane) to afford the methylacetal (3.35 g, 66%).

This product (3.35 g 5.7 mmol) was dissolved in methanol (25 ml) and asolution of mercuric oxide (56 mg, 0.26 mmol) water (3.5 ml) andconcentrated sulphuric acid (0.75 ml, 14 mmol)was added dropwise at 20°C. The mixture was stirred at 20° C. for 2 h., water was added and themixture was extracted with dichloromethane (3×100 ml). The combinedorganic layers were washed with sodium bicarbonate solution (100 ml),dried (MgSO₄) and evaporated to give the title ketone (3.2 g)) pure bynmr + tlc.

EXAMPLE 1 VS 54396 (=Z-isomer)

23-Oxo-25(S)-t-butyl milbemycin X (50 mg, 0.09 mmol) was dissolved inmethanol (5 ml). A solution of methoxylamine hydrochloride (50 mg, 0.60mmol) in water (2 ml) was added and the mixture stirred at roomtemperature (1h). The reaction mixture was concentrated and then treatedwith water (30 ml) and extracted with ether (3×15 ml). The combinedethereal extracts were dried (MgSO₄) and evaporated. The 1:1 mixture* ofZ and E oximes was separated by silica gel preparative thin layerchromatography with hexane - ethyl acetate, 1:1 as eluant.

The 23(Z)-methoxyimino-25(S)-t-butyl milbemycin X was obtained as awhite solid (yield 16 mg) ^(M/) Z (FAB ^(Na+/Noba)) 622 [MNa]⁺ 50%(relative intensity). Hplc retention time =7.7 min.

The 23(E)-methoxyimino-25(S)-t-butyl milbemycin X was obtained as awhite solid (yield 16 mg) ^(M/) Z (FAB ^(Na+/Noba)) 622 [MNa]⁺ 25%(relative intensity). Hplc retention time =7.9 min.

Hplc conditions: Dynamax C18 column (25 cm×4.6 mm id) eluted withmethanol--water , 9: 1 at 1 ml/min monitored at 245 nm. * Ratio isdependent upon the pH of the reaction mixture.

EXAMPLE 2 VS 55759 (=E-isomer)

To a solution of 23-oxo-25(S)-t-butyl milbemycin X (60 mg 0.1 mmol) andsodium acetate (300 mg, 2.2 mmol) in methanol (3 ml) was added0-t-butylhydroxylamine hydrochloride (50 mg, 0.4 mmol). The mixture wasstirred at 20° C. for 1 h., water (10 ml) was added and the wholemixture extracted with dichloromethane (3×15 ml). The combined organicextracts were washed with water, dried (MgSO₄) and evaporated todryness. Purification by preparative t.l.c.,(silica taper plate(Analtech^(R)) eluted with ethyl acetate/hexane 2:5) yielded a 4:1mixture of E:Z oxime isomers (54 mg). Treatment of a portion of thismixture (30 mg) with methanol (2 ml) and hydrochloric acid (1M, 0.2 ml)gave a 1:1 E:Z ratio of oxime isomers. The two isomers were separated bypreparative t.l.c. (silica taperplate eluted four times withchloroform).

23(Z)-t-butyloxyimino-25(S)-t-butyl milbemycin X:

tlc R_(f) =0.5 (Silica eluted three times with chloroform containing1.5% ethanol) m/z (FAB Na⁺ /Noba) (relative intensity) 664 [MNa]⁺ (95%)

23(E)-t-butyloxyimino-25(S)-t-butyl milbemycin X: tlc R_(f) =0.45(Silica eluted three times with chloroform containing 1.5% ethanol) m/z(FAB Na⁺ /Noba) (relative intensity) 664 [MNa]⁺ (95%).

Hplc retention times: Dynamax 60A Silica column (25 cm×4.6 mm id) elutedwith chloroform/methanol 99:1 at 1 ml/min monitored at 245 nm.

Retention time 23-Z isomer=14.2 min.

Retention time 23-E isomer=15.4 min.

Efficacy of VS-54936 and VS-55759 against Dirofilaria immitis in dogs

Method

Twenty-one adult male and 21 adult female beagle dogs were used. Thedogs weighed between 7.8 and 11.3 kg, and their ages ranged from 12 to13 months. Blood from all of the 42 dogs was collected prior toinitiation of the study and at 4.5 months postinfection and examined bythe modified Knott's method to ascertain that the dogs were not infectedwith either D. immitis or Dipetalonema reconditum.

On day 0 each dog was given 50 infective larvae of p. immitis asdescribed by McCall (J. Georgia Entomol. Soc. 16(2), 1981). The dogswere weighed on Day 23 and allocated to treatment groups. Six replicatesof 7 dogs each were formed by ranking dogs by weight within the sex.Within each replicate, the dogs were randomly allocated to 1 of 7treatment groups using a table of random numbers (Bhattacharyya, G.K.and R.A. Johnson, Statistical Concepts and Methods, John Wiley and Sons,N.Y., 1977, Table 14, pp. 623-624), such that 7 groups of 6 dogs each (3males and 3 females) were formed.

On Day 30 i.e., 30 days post-infection, each dog was dosed orally with agelatin capsule containing the compound under test.

All dogs were killed on the same day at 149 days after inoculation ofinfective larvae of D. immitis.

The pleural and peritoneal cavities were examined for immature andmature adult worms of D. immitis, and the anterior and posterior venaecavae and the azygous vein were ligated before removal of the heart andlungs. The precava, right atrium, right ventricle, and pulmonaryarteries (including those coursing through the lungs) were dissected andexamined for worms. The worms from each dog were recorded as either deador alive and either immature or mature.

Results

The groups treated at 0.01 mg/kg with VS 54936 or VS 55759, all dogswere free of worms. No adverse reactions associated with treatment wereobserved for any treated animal in any of the treatment groups.

Efficacy of VS-55759 and VS-54936 for control of Demodex canis InfestingDogs

Method

Fifteen dogs of beagle breeding with natural Demodex infestations werepreconditioned. Preconditioning included worming and vaccinations fordistemper, hepatitis, leptospirosis, parvovirus and parainfluenza.

The fifteen dogs were subdivided into five groups of three dogs pertreatment group. The dogs were housed one per pen and group isolated toavoid cross-contamination. Food and water was available ad libitum.

The compounds under test were administered by subcutaneous injection onthree occasions at 0.7 and 14 days.

Efficacy was determined by skin scraping and clinical observations. Theskin was scraped and scrapings examined to determine Demodex mitepopulations. A small amount of mineral oil was applied to an infestedarea on the belly, legs, feet, or face, and approximately 12 squarecentimeters of skin was scraped with a scalpel. The skin was scrapeduntil blood was observed. The total amount of material collected wasdiluted with mineral oil and microscopically examined for mites andeggs. Observations were mapped on each quarter of each dog at eachobservation period on days 21, 28, 35 and 42.

Results

Dogs treated with 3×200 mg of VS 54396 and VS 55759 were clear of mitesat day 35 and day 21 respectively and remained clear until the end ofthe trial.

No adverse reactions to treatment were noted.

I claim:
 1. A pharmaceutical composition for use in treatment orprophylaxis of endo- and ectoparasitic infestations in dogs, whichcomprises a compound of formula (I): ##STR4## wherein R¹ is selectedfrom the group consisting of hydrogen, hydroxy, and protected hydroxy;R²is selected from the group consisting of alkoxy, hydroxy, protectedhydroxy, oxo, oximino, and O-substituted oximino; R³ is selected fromthe group consisting of hydrogen, hydroxy, protected hydroxy,4'-(α-L-oleandrosyl)-α-L-oleandrosyloxy,4'-(α-L-oleandrosyl)-α-L-oleandrosyloxy, wherein the terminal hydroxygroup is protected, α-L-oleandrosyloxy, and α-L-oleandrosyloxy whereinthe terminal hydroxy group is protected; R⁴, R⁵, R⁶, and R⁷ areindependently selected from the group consisting of hydrogen and anorganic radical; R⁸ is selected from the group consisting of amino,substituted amino, imino, and substituted imino; provided thatA) if R²is hydroxy or protected hydroxy; R³ is hydrogen; R⁴ is hydrogen ormethyl; R⁵ is hydrogen or methyl; and R⁸ is imino, oxyimino,O-substituted oxyimino, hydrazone, N-substituted hydrazone,semicarbazone, or N-substituted semicarbazone;then i) R¹ is hydroxy orprotected hydroxy; and further provided that (a) when R⁶ is hydrogen, R⁷is not cis-C(CH₃)═CHR^(n), where R^(n) is selected from the groupconsisting of methyl, ethyl, and isopropyl; (b) when R⁷ is hydrogen, R⁶is not cis-C(CH₃)═CHR^(n), where R^(n) is selected from the groupconsisting of methyl, ethyl, and isopropyl; (c) when R⁶ is hydrogen, R⁷is not ##STR5## where R^(n) is selected from the group consisting ofmethyl, ethyl, and isopropyl; (d) when R⁷ is hydrogen, R⁶ is not##STR6## where R^(n) is selected from the group consisting of methyl,ethyl, and isopropyl; B) if R² is hydroxy or protected hydroxy; R³ ishydrogen; R⁴ is hydrogen or methyl; R⁵ is hydrogen or methyl; R⁶ ishydrogen, isopropyl, or sec-butyl; R⁷ is hydrogen, isopropyl, orsec-butyl; and R⁸ is imino, oxyimino, O-substituted oxyimino, hydrazone,or N-substituted hydrazone;then i) R¹ is hydroxy or protected hydroxy;C) if R² is oxo, hydroxy, substituted hydroxy having up to 25 carbonatoms; R³ is hydroxy; R⁴ is methyl; R⁵ is hydrogen; R⁶ is hydrogen; R⁷is cis-C(CH₃)═CHR^(o), where R^(o) is selected from the group consistingof methyl, ethyl, and isopropyl; and R⁸ is --C═NOR^(p), where R^(p) isselected from the group consisting of hydrogen and a C₁₋₈ alkylgroup:then i) R¹ is hydroxy or protected hydroxy;said compound offormula (I) being the E or Z isomer, or a mixture thereof.
 2. Acomposition according to claim 1, in the form of an oral formulation. 3.A composition according to claim 1, in the form of an injectableformulation.
 4. A composition according to claim 1, in the form of apour-on formulation.
 5. A method for the treatment or prophylaxis ofendo- and ectoparasitic infestations in dogs, which method comprises theadministration to a dog in need thereof of an effective amount of acompound of formula (I): ##STR7## wherein R¹ is selected from the groupconsisting of hydrogen, hydroxy, and protected hydroxy;R² is selectedfrom the group consisting of alkoxy, hydroxy, protected hydroxy, oxo,oximino, and O-substituted oximino; R³ is selected from the groupconsisting of hydrogen, hydroxy, protected hydroxy,4'-(α-L-oleandrosyl)-α-L-oleandrosyloxy,4'-(α-L-oleandroyl)-α-L-oleandrosyloxy wherein the terminal hydroxygroup is protected, α-L-oleandrosyloxy, and α-L-oleandrosyloxy whereinthe terminal hydroxy group is protected; R⁴, R⁵, R⁶, and R⁷ areindependently selected from the group consisting of hydrogen and anorganic radical; R⁸ is selected from the group consisting of amino,substituted amino, imino, and substituted imino; provided thatA) if R²is hydroxy or protected hydroxy; R³ is hydrogen; R⁴ is hydrogen ormethyl; R⁵ is hydrogen or methyl; and R⁸ is imino, oxyimino,O-substituted oxyimino, hydrazone, N-substituted hydrazone,semicarbazone, or N-substituted semicarbazone;then i) R¹ is hydroxy orprotected hydroxy; and further provided that (a) when R⁶ is hydrogen, R⁷is not cis-C(CH₃)═CHR^(n), where R^(n) is selected from the groupconsisting of methyl, ethyl, and isopropyl; (b) when R⁷ is hydrogen, R⁶is not cis-C(CH₃)═CHR^(n), where R^(n) is selected from the groupconsisting of methyl, ethyl, and isopropyl; (c) when R⁶ is hydrogen, R⁷is not ##STR8## where R^(n) is selected from the group consisting ofmethyl, ethyl, and isopropyl; (d) when R⁷ is hydrogen, R⁶ is not##STR9## where R^(n) is selected from the group consisting of methyl,ethyl and isopropyl; B) if R² is hydroxy or protected hydroxy; R³ ishydrogen; R⁴ is hydrogen or methyl; R⁵ is hydrogen or methyl; R⁶ ishydrogen, isopropyl, or sec-butyl; R⁷ is hydrogen, isopropyl, orsec-butyl; and R⁸ is imino, oxyimino, O-substituted oxyimino, hydrazone,or N-substituted hydrazone;then i) R¹ is hydroxy or protected hydroxy;C) if R² is oxo, hydroxy, substituted hydroxy having up to 25 carbonatoms; R³ is hydroxy; R⁴ is methyl; R⁵ is hydrogen; R⁶ is hydrogen; R⁷is cis-C(CH₃)═CHR^(o), where R^(o) is selected from the group consistingof methyl, ethyl, and isopropyl; and R⁸ is --C═NOR^(p), where R^(p) isselected from the group consisting of hydrogen and a C₁₋₈ alkylgroup;then i) R¹ is hydroxy or protected hydroxy;said compound offormula (I) being the E or Z isomer, or a mixture thereof.
 6. A methodaccording to claim 5, wherein said method is for the treatment ofendoparasitic and ectoparasitic infestations selected from the groupconsisting of roundworm, hookworm, whipworm and/or demodectic mange. 7.A method according to claim 5, wherein said method is for theprophylaxis of heartworm infestation.
 8. A method according to claim 5,wherein R¹, R³, R⁴, R⁵ and R⁶ are hydrogen, R² is hydroxy, R⁷ is t-butyland R⁸ is methoxyimino or t-butyloxyimino, the E- or Z-isomer or amixture thereof.